2.1 PROTEIN PHOSPHORYLATION AND SIGNAL TRANSDUCTION
Cells rely, to a great extent, on extracellular molecules as a means by which to receive stimuli from their immediate environment. These extracellular signals are essential for the correct regulation of such diverse cellular processes as differentiation, contractility, secretion, cell division, contact inhibition, and metabolism. The extracellular molecules, which can include, for example, hormones, growth factors, lymphokines, or neurotransmitters, act as ligands that bind to specific cell surface receptors. The binding of these ligands to their receptors triggers a cascade of reactions that brings about both the amplification of the original stimulus and the coordinate regulation of the separate cellular processes mentioned above. In addition to normal cellular processes, receptors and their extracellular ligands may be involved in abnormal or potentially deleterious processes such as virus-receptor interaction, inflammation, and cellular transformation to a cancerous state.
A central feature of this process, referred to as signal transduction (for recent reviews, see Posada, J. and Cooper, J. A., 1992, Mol. Biol. Cell 3:583-592; Hardie, D. G., 1990, Symp. Soc. Exp. Biol. 44:241-255), is the reversible phosphorylation of certain proteins. The phosphorylation or dephosphorylation of amino acid residues triggers conformational changes in regulated proteins that alter their biological properties. Proteins are phosphorylated by protein kinases and are dephosphorylated by protein phosphatases. Protein kinases and phosphatases are classified according to the amino acid residues they act on, with one class being serine-threonine kinases and phosphatases (reviewed in Scott, J. D. and Soderling, T. R., 1992, 2:289-295), which act on serine and threonine residues, and the other class being the tyrosine kinases and phosphatases (reviewed in Fischer, E. H. et al., 1991 Science 253:401-406; Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212), which act on tyrosine residues. The protein kinases and phosphatases may be further defined as being receptors, i.e., the enzymes are an integral part of a transmembrane, ligand-binding molecule, or as non-receptors, meaning they respond to an extracellular molecule indirectly by being acted upon by a ligand-bound receptor. Phosphorylation is a dynamic process involving competing phosphorylation and dephosphorylation reactions, and the level of phosphorylation at any given instant reflects the relative activities, at that instant, of the protein kinases and phosphatases that catalyze these reactions.
While the majority of protein phosphorylation occurs at serine and threonine amino acid residues, phosphorylation at tyrosine residues also occurs, and has begun to attract a great deal of interest since the discovery that many oncogene products and growth factor receptors possess intrinsic protein tyrosine kinase activity. The importance of protein tyrosine phosphorylation in growth factor signal transduction, cell cycle progression and neoplastic transformation is now well established (Cantley, L. C. et al., 1991, Cell 64:281-302; Hunter T., 1991, Cell 64:249-270; Nurse, 1990, Nature 344:503-508; Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212). Subversion of normal growth control pathways leading to oncogenesis has been shown to be caused by activation or overexpression of protein tyrosine kinases which constitute a large group of dominant oncogenic proteins (reviewed in Hunter, T., 1991, Cell 64:249-270).
2.2 PROTEIN TYROSINE KINASES
Protein tyrosine kinases comprise a large family of proteins, including many growth factor receptors and potential oncogenes, which share ancestry with, but nonetheless differ from, serine/threonine-specific protein kinases (Hanks et al., 1988, Science 241:42-52).
Receptor-type protein tyrosine kinases having a transmembrane topology have been studied extensively. The binding of a specific ligand to the extracellular domain of a receptor protein tyrosine kinase is thought to induce receptor dimerization and phosphorylation of their own tyrosine residues. Individual phosphotyrosine residues of the cytoplasmic domains of receptors may serve as specific binding sites that interact with a host of cytoplasmic signalling molecules, thereby activating various signal transduction pathways (Ullrich, A. and Schlessinger, J., 1990, Cell 61:203-212).
The intracellular, cytoplasmic, non-receptor protein tyrosine kinases, may be broadly defined as those protein tyrosine kinases which do not contain a hydrophobic, transmembrane domain. Within this broad classification, one can divide the known cytoplasmic protein tyrosine kinases into eleven distinct morphotypes, including the SRC family (Martinez, R. et al., 1987, Science 237:411-414; Sukegawa, J. et al., 1987, Mol. Cell. Biol., 7:41-47; Yamanishi, Y. et al., 1987, 7:237-243; Marth, J. D. et al., 1985, Cell 43:393-404; Dymecki, S.M. et al., 1990, Science 247:332-336), the FES family (Ruebroek, A. J. M. et al., 1985, EMBO J. 4:2897-2903; Hao, Q. et al., 1989, Mol. Cell. Biol. 9:1587-1593), the ABL family (Shtivelman, E. et al., 1986, Cell 47:277-284; Kruh, G. D. et al., 1986, Science 234:1545-1548), the Za.sub.p 70 family and the JAK family. While distinct in their overall molecular structure, each of the members of these morphotypic families of cytoplasmic protein tyrosine kinases share non-catalytic domains in addition to sharing their catalytic kinase domains. Such non-catalytic domain are the SH2 (SRC homology domain 2; Sadowski, I. et al., Mol. Cell. Biol. 6: 4396-4408; Koch, C. A. et al., 1991, Science 252:668-674) domains and SH3 domains (Mayer, B. J. et al., 1988, Nature 332:269-272). Such non-catalytic domains are thought to be important in the regulation of protein-protein interactions during signal transduction (Pawson, T. and Gish, G., 1992, Cell 71:359-362).
While the metabolic roles of cytoplasmic protein tyrosine kinases are less well understood than that of the receptor-type protein tyrosine kinases, significant progress has been made in elucidating some of the processes in which this class of molecules is involved. For example, members of the src family, lck and fyn, have been shown to interact with CD4/CD8 and the T cell receptor complex, and are thus implicated in T cell activation, (Veillette, A. and Davidson, D., 1992, TIG 8:61-66), certain cytoplasmic protein tyrosine kinases have been linked to certain phases of the cell cycle (Morgan, D. O. et al., 1989, Cell 57: 775-786; Kipreos, E. T. et al., 1990, Science 248:. 217-220; Weaver et al., 1991, Mol. Cell. Biol. 11:4415-4422), and cytoplasmic protein tyrosine kinases have been implicated in neuronal development (Maness, P., 1992, Dev. Neurosci 14:257-270). Deregulation of kinase activity through mutation or overexpression is a well-established mechanism underlying cell transformation (Hunter et al., 1985, supra; Ullrich et al., supra).
2.3 ADAPTOR PROTEINS
Adaptor proteins are intracellular proteins having characteristic conserved peptide domains (SH2 and/or SH3 domains, as described below) which are critical to the signal transduction pathway. Such adaptor proteins serve to link protein tyrosine kinases, especially receptor-type protein tyrosine kinases to downstream intracellular signalling pathways such as the RAS signalling pathway. It is thought that such adaptor proteins may be involved in targeting signal transduction proteins to the correct site in the plasma membrane or subcellular compartments, and may also be involved in the regulation of protein movement within the cell.
Such adaptor proteins are among the protein substrates of the receptor-type protein tyrosine kinases, and have in common one or two copies of an approximately 100 amino acid long motif. Because this motif was originally identified in c-Src-like cytoplasmic, non-receptor tyrosine kinases it is referred to as a Src homology 2 (SH2) domain. SH2-containing polypeptides may otherwise, however, be structurally and functionally distinct from one another (Koch, C. A. et al., 1991, Science 252:668-674). SH2 domains directly recognize phosphorylated tyrosine amino acid residues. The peptide domains also have independent sites for the recognition of amino acid residues surrounding the phosphotyrosine residue(s).
When a receptor protein tyrosine kinase binds an extracellular ligand, receptor dimerization is induced, which, in turn, leads to intermolecular autophosphorylation of the dimerized kinases (Schlessinger, J. and Ullrich, A., 1992, Neuron 9: 383-391). Receptor phosphorylation, therefore, creates SH2-binding sites, to which an adaptor protein may bind.
In addition to SH2 peptide domains, many of the adaptor proteins involved in signal transduction contain a second conserved motif of 50-75 amino acids residues, the SH3 domain (Schlessinger, J. and Ullrich, A., 1992, Neuron 9:383-391; Pawson, T. and Gish, G. D., 1992, Cell 72:359-362; Mayer, B. J. and Baltimore, D., 1993, Trends in Cell Biol. 3 8-13; Mayer, B. J. et al., 1988, Nature 352:272-275). Much less is known about the biological role of the SH3 domain than is known about the role of SH2. The current view is that SH3 domains function, in part, as protein-binding domains that act to link signals transmitted from the cell surface to downstream effector genes such as ras (Pawson, T. and Schlessinger, J., 1993 Current Biology, 3:434-442).
2.4 G-PROTEINS AND SIGNAL TRANSDUCTION
Guanine-nucleotide-binding proteins, (G-proteins; Simon, M. I. et al., 1991, Science 252:802-808; Kaziro, Y. et al., 1991, Ann. Rev. Biochem. 60:349-400) such as Ras (for review, see Lowy, D. R. and Willumsen, B. M., 1993, Ann Rev. Biochem. 62:851-891), play an essential role in the transmission of mitogenic signals from receptor tyrosine kinases. Taking Ras as an example, the activation of receptor tyrosine kinases by ligand binding results in the accumulation of the active GTP bound form of the Ras molecule (Gibbs, J. B. et al., 1990, J. Biol. Chem. 265:20437-2044; Satoh, T. et al., 1990, Proc. NaTl. Acad. Sci. USA 87:5993-5997; Li, B.-Q. et al., 1992, Science 256:1456-1459; Buday, L. and Downward, J., 1993, Mol. Cell. Biol. 13:1903-1910; Medema, R. H. et al., 1993, Mol. Cell. Biol. 13:155-162). Ras activation is also required for transformation by viral oncogenic tyrosine kinases (Smith, M. R. et al., 1986, Nature 320:540-43).
Ras activity is regulated by the opposing actions of the GTPase-activating proteins (GAPs) and guanine nucleotide exchange factors, with GAPs stimulating the slow intrinsic rate of GTP hydrolysis on Ras and exchange factors stimulating the basal rate of exchange of GDP for GTP on Ras. Thus, GAPs act as negative regulators of Ras function, while exchange factors act as Ras activators.
Recently, a direct link between activated receptor tyrosine kinases and Ras was established with the finding that the mammalian GRB-2 protein, a 26 kilodalton protein comprised of a single SH2 and two SH3 domains (Lowenstein, E. J. et al., 1992, Cell 70:431-442), directly couples receptor tyrosine kinases to the Ras exchange factor Sos in mammals and Drosophila (Buday, L. and Downward, J., 1993, Cell 73:611-620; Egan, S. E. et al., 1993, Nature 363:45-51; Li, N. et al., 1993, Nature 363:85-87; Gale, N. W. et al., 1993, Nature 363:88-92; Rozakis-Adcock et al., 1993, Nature 363:83-85; Chardin, P. et al., 1993, Science 260:1338-1343; Oliver, J. P. et al., Cell 73:179-191; Simon, M. A. et al., 1993, Cell 73:169-177). The GRB-2 SH2 domain binds to specific tyrosine phosphorylated sequences in receptor tyrosine kinases while the GRB-2 SH3 domains bind to proline-rich sequences present in the Sos exchange factor. Binding of GRB-2 to the receptor kinases, therefore, allows for the recruitment of Sos to the plasma membrane, where Ras is located (Schlessinger, J., 1993, TIBS 18:273-275).
Grb2 has been shown to be associated with CSF-1 receptor (vanderGeer and Hunter, 1993, EMBO J. 12(13):5161-5172), PDGF receptor (Li et al., 1994, MCB 14(1):509-517), EGF-R (Matuoka et al., 1993, EMBO J. 12(9):3467-3475; Lowenstein et al., 1992, Cell 70:431-442) and Fak (Schlaepfer et al., 1994, Nature 372:786-791), amongst other proteins.
2.5 CELL PROLIFERATIVE DISORDERS
Growth factors and their receptors are crucial for normal development but can also act as oncogenes leading to cell transformation, oncogenesis, and cell proliferative disorders, including cancer. Activation of the oncogenic potential of normal cellular proteins such as protein tyrosine kinases may, for example, occur by alteration of the proteins' corresponding enzymatic activities, their inappropriate binding to other cellular components, or both.
Taking as an example Philadelphia chromosome-positive human leukemias, it is known that the BCR-ABL oncoprotein is involved in the pathenogenesis of such leukemias. BCR-ABL exhibits deregulated tyrosine kinase activity. It has recently been demonstrated (Pendergast, A. M. et al., 1993, Cell 75:175-185) that BCR-ABL binds the SH2/SH3 domain-containing GRB-2 adaptor protein. Further, it has been demonstrated that BCR-ABL/GRB-2 binding is mediated by the direct interaction the GRB-2 SH2 domain and a tyrosine-phosphorylated region of the BCR-ABL protein, and that this interaction is required for the activation of the Ras signaling pathway.
Thus, there are multiple events which occur along a signal transduction pathway which appear to be required for the ultimate appearance of a cell proliferative disorder such as the form of leukemia described above. One approach to the treatment of oncogenenic, cell proliferative disorders would be to attempt to "short circuit" abnormal signal transduction events which contribute to the appearance of such disorders, by interfering with one or more of these requisite events.
The amelioration of an abnormal kinase activity may be interfered with by targeting and directly inhibiting the enzymatic activity of the kinase involved in the cell proliferative disorder. It has been proposed that certain compounds may have such anti-tyrosine kinase activity. See, for example, Levitzki and Gazit, 1995, Science 267:1782-1788, wherein certain quinazoline derivatives are proposed to directly inhibit receptor tyrosine kinase enzymatic activity.
In instances wherein the signal transduction event of interest involves an adaptor protein/protein tyrosine kinase interaction, the inhibition of such interactions may lead to the amelioration of cell proliferative disorder symptoms. The utility of this approach has been demonstrated using expression of signaling incompetent proteins in cells. For example, cells expressing a mutant form of Bcr-Ab1 which lacks the tyrosine residue necessary for binding of the GrB2 SH2 domain and is thus signaling incompetent no longer exhibits a transformed phenotype (RER) (Pendergast et al., supra). To date, however, no such inhibitor of adaptor protein/protein tyrosine kinase interactions has been identified.